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CytoJournal 2022Liquid-based cytology (LBC) is a monolayer slide preparation technology that has outperformed conventional Pap smears because of improved fixation, decreased obscuring... (Review)
Review
Liquid-based cytology (LBC) is a monolayer slide preparation technology that has outperformed conventional Pap smears because of improved fixation, decreased obscuring factors, and standardized cell transfer. In LBC, samples are collected by completely immersing the sampling device into the company vial containing preservative fluid, whereby the cells are preserved and fixed simultaneously unlike conventional smears where the sample is smeared onto the glass slide and fixed separately. To date, two major liquid-based preparation methods are known - ThinPrep and SurePath. These two methods are different in their principles of cell harvesting but produce similar preparations. SurePath works on the principle of density gradient sedimentation. In this, a sample is vortexed and strained to break the mucus and large cell groups and then is treated through a density gradient centrifugation process to remove blood and debris. The cell pellet is resuspended and is allowed to sediment onto a glass slide. This is followed by staining on the PrepStain instrument. Government Medical College and Hospital, Nagpur, India, uses the SurePath method which was approved by FDA in the USA in 1999. Our institution uses Rovers Cervex-Brush to collect the cells from the transformation zone. This chapter describes the principle of SurePath and the processing of cervicovaginal specimen using the fully automated system in the laboratory.
PubMed: 35928530
DOI: 10.25259/CMAS_03_16_2021 -
New Biotechnology Mar 2020When dealing with pre-analytics for tissues, it is often that case that tissue heterogeneity, and particularly tumor heterogeneity, is not taken into account as a... (Review)
Review
When dealing with pre-analytics for tissues, it is often that case that tissue heterogeneity, and particularly tumor heterogeneity, is not taken into account as a preliminary condition for obtaining reproducible results in molecular analysis at the diagnostics and clinical research levels. It is well known that when sampling tumor tissues in different areas, for example the border or the central area of the tumor, different genes are expressed and, due to polyclonality in most tumors, different areas can have different DNA and epigenetic alterations. For this reason, it is extremely important to establish and standardize specific tissue sampling protocols for molecular extraction as well as in situ molecular methods. A correct approach to heterogeneity is the basis for a more reproducible and exchangeable type of molecular analysis that can provide useful information at the prognostic and predictive levels. Heterogeneity should also be taken into consideration during cancer treatment, since therapy modifies the clonal composition of tumors. Here, the different types of tumor heterogeneity and the improper pre-analytical conditions in tissue processing that can generate heterogeneous artefacts are described.
Topics: Genetic Heterogeneity; Humans; Neoplasms; Organ Preservation; Pre-Analytical Phase; Tissue Fixation
PubMed: 31580921
DOI: 10.1016/j.nbt.2019.09.007 -
Nucleic Acids Research Aug 2023In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day... (Review)
Review
In the late 19th century, formalin fixation with paraffin-embedding (FFPE) of tissues was developed as a fixation and conservation method and is still used to this day in routine clinical and pathological practice. The implementation of state-of-the-art nucleic acid sequencing technologies has sparked much interest for using historical FFPE samples stored in biobanks as they hold promise in extracting new information from these valuable samples. However, formalin fixation chemically modifies DNA, which potentially leads to incorrect sequences or misinterpretations in downstream processing and data analysis. Many publications have concentrated on one type of DNA damage, but few have addressed the complete spectrum of FFPE-DNA damage. Here, we review mitigation strategies in (I) pre-analytical sample quality control, (II) DNA repair treatments, (III) analytical sample preparation and (IV) bioinformatic analysis of FFPE-DNA. We then provide recommendations that are tested and illustrated with DNA from 13-year-old liver specimens, one FFPE preserved and one fresh frozen, applying target-enriched sequencing. Thus, we show how DNA damage can be compensated, even when using low quantities (50 ng) of fragmented FFPE-DNA (DNA integrity number 2.0) that cannot be amplified well (Q129 bp/Q41 bp = 5%). Finally, we provide a checklist called 'ERROR-FFPE-DNA' that summarises recommendations for the minimal information in publications required for assessing fitness-for-purpose and inter-study comparison when using FFPE samples.
Topics: DNA; Formaldehyde; Paraffin Embedding; Sequence Analysis, DNA; Tissue Fixation
PubMed: 37351572
DOI: 10.1093/nar/gkad519 -
Applied and Environmental Microbiology Jun 2022Soil nitrogen (N) transformations constrain terrestrial net primary productivity and are driven by the activity of soil microorganisms. Free-living N fixation (FLNF) is...
Soil nitrogen (N) transformations constrain terrestrial net primary productivity and are driven by the activity of soil microorganisms. Free-living N fixation (FLNF) is an important soil N transformation and key N input to terrestrial systems, but the forms of N contributed to soil by FLNF are poorly understood. To address this knowledge gap, a focus on microorganisms and microbial scale processes is needed that links N-fixing bacteria and their contributed N sources to FLNF process rates. However, studying the activity of soil microorganisms poses inherent challenges, including differences in sampling scale between microorganism and process rates, which can be addressed with culture-based studies and an emphasis on microbial-scale measurements. Culture conditions can differ significantly from soil conditions, so it also important that such studies include multiple culture conditions like liquid and solid media as proxies for soil environments like soil pore water and soil aggregate surfaces. Here we characterized extracellular N-containing metabolites produced by two common, diazotrophic soil bacteria in liquid and solid media, with or without N, across two sampling scales (bulk via GC-MS and spatially resolved via MALDI mass spec imaging). We found extracellular production of inorganic and organic N during FLNF, indicating terrestrial N contributions from FLNF occur in multiple forms not only as ammonium as previously thought. Extracellular metabolite profiles differed between liquid and solid media supporting previous work indicating environmental structure influences microbial function. Metabolite profiles also differed between sampling scales underscoring the need to quantify microbial scale conditions to accurately interpret microbial function. Free-living nitrogen-fixing bacteria contribute significantly to terrestrial nitrogen availability; however, the forms of nitrogen contributed by this process are poorly understood. This is in part because of inherent challenges to studying soil microorganisms , such as vast differences in scale between microorganism and ecosystem and complexities of the soil system (e.g., opacity, chemical complexity). Thus, upscaling important ecosystem processes driven by soil microorganisms, like free-living nitrogen fixation, requires microbial-scale measurements in controlled systems. Our work generated bulk and spatially resolved measurements of nitrogen released during free-living nitrogen fixation under two contrasting growth conditions analogous to soil pores and aggregates. This work allowed us to determine that diverse forms of nitrogen are likely contributed to terrestrial systems by free-living nitrogen bacteria. We also demonstrated that microbial habitat (e.g., liquid versus solid media) alters microbial activity and that measurement of microbial activity is altered by sampling scale (e.g., bulk versus spatially resolved) highlighting the critical importance of quantifying microbial-scale processes to upscaling of ecosystem function.
Topics: Bacteria; Ecosystem; Metabolome; Nitrogen; Nitrogen Fixation; Soil; Soil Microbiology
PubMed: 35652664
DOI: 10.1128/aem.00505-22 -
Sensors (Basel, Switzerland) Nov 2022Eye tracking is a technology aimed at understanding the direction of the human gaze. Event detection is a process of detecting and classifying eye movements that are... (Review)
Review
Eye tracking is a technology aimed at understanding the direction of the human gaze. Event detection is a process of detecting and classifying eye movements that are divided into several types. Nowadays, event detection is almost exclusively done by applying a detection algorithm to the raw recorded eye-tracking data. However, due to the lack of a standard procedure for how to perform evaluations, evaluating and comparing various detection algorithms in eye-tracking signals is very challenging. In this paper, we used data from a high-speed eye-tracker SMI HiSpeed 1250 system and compared event detection performance. The evaluation focused on fixations, saccades and post-saccadic oscillation classification. It used sample-by-sample comparisons to compare the algorithms and inter-agreement between algorithms and human coders. The impact of varying threshold values on threshold-based algorithms was examined and the optimum threshold values were determined. This evaluation differed from previous evaluations by using the same dataset to evaluate the event detection algorithms and human coders. We evaluated and compared the different algorithms from threshold-based, machine learning-based and deep learning event detection algorithms. The evaluation results show that all methods perform well for fixation and saccade detection; however, there are substantial differences in classification results. Generally, CNN (Convolutional Neural Network) and RF (Random Forest) algorithms outperform threshold-based methods.
Topics: Humans; Eye Movements; Algorithms; Saccades; Neural Networks, Computer; Machine Learning
PubMed: 36433407
DOI: 10.3390/s22228810 -
Veterinary Sciences Jan 2022Cetacean brain sampling may be an arduous task due to the difficulty of collecting and histologically preparing such rare and large specimens. Thus, one of the main...
Cetacean brain sampling may be an arduous task due to the difficulty of collecting and histologically preparing such rare and large specimens. Thus, one of the main challenges of working with cetaceans' brains is to establish a valid methodology for an optimal manipulation and fixation of the brain tissue, which allows the samples to be viable for neuroanatomical and neuropathological studies. With this in view, we validated a methodology in order to preserve the quality of such large brains (neuroanatomy/neuropathology) and at the same time to obtain fresh brain samples for toxicological, virological, and microbiological analysis (neuropathology). A fixation protocol adapted to brains, of equal or even three times the size of human brains, was studied and tested. Finally, we investigated the usefulness of a panel of 20 antibodies (neuromarkers) associated with the normal structure and function of the brain, pathogens, age-related, and/or functional variations. The sampling protocol and some of the 20 neuromarkers have been thought to explore neurodegenerative diseases in these long-lived animals. To conclude, many of the typical measures used to evaluate neuropathological changes do not tell us if meaningful cellular changes have occurred. Having a wide panel of antibodies and histochemical techniques available allows for delving into the specific behavior of the neuronal population of the brain nuclei and to get a "fingerprint" of their real status.
PubMed: 35202291
DOI: 10.3390/vetsci9020038 -
Biopreservation and Biobanking Apr 2023This work investigates whether changes in a biospecimen's molecular composition from formaldehyde fixation drive changes in the mid infrared (MID-IR) spectrum. Our...
This work investigates whether changes in a biospecimen's molecular composition from formaldehyde fixation drive changes in the mid infrared (MID-IR) spectrum. Our ultimate goal was to develop an analytical metrology that could be used to accurately determine the fixation time of a tissue sample as a surrogate to overall tissue quality. Multiple unstained formalin-fixed paraffin-embedded tissue samples were scanned with an MID-IR microscope to identify a molecular fingerprint of formaldehyde fixation. The fixation specific patterns were then mined to develop a predictive model. A multiple tissue experiment using greater than 100 samples was designed to train the algorithm and validate the accuracy of predicting fixation status. We present data that formaldehyde crosslinking results in alterations to multiple bands of the MID-IR spectra. The impact was most dramatic in the Amide I band, which is sensitive to the conformational state of proteins. The spectroscopic fixation signature was used to train a machine-learning model that could predict fixation time of unknown tissues with an average accuracy of 1.4 hours. Results were validated by histological stain quality for bcl-2, FOXP3, and ki-67. Further, two-dimensional imaging was used to visualize the spatial dependence of fixation, as demonstrated by multiple features in the tissue's vibrational spectra. This work demonstrates that it is possible to predict the fixation status of tissues for which the preanalytics are unknown. This novel capability could help standardize clinical tissue diagnostics and ensure every patient gets the absolutely best treatment based on the highest quality tissue sample.
Topics: Humans; Tissue Fixation; Spectrophotometry, Infrared; Formaldehyde; Proteins; Machine Learning; Paraffin Embedding
PubMed: 36516138
DOI: 10.1089/bio.2022.0108 -
Cureus Feb 2023Silicone implants are one of the most widely used implants for facial augmentation, especially in the chin, mandibular angle, and malar area, utilizing different... (Review)
Review
Silicone implants are one of the most widely used implants for facial augmentation, especially in the chin, mandibular angle, and malar area, utilizing different surgical approaches. Despite their various advantages, many complications have also been reported, including hematoma, infection, bone resorption, numbness, displacement, and asymmetry. This study aims to evaluate the need for facial-implant fixation and compare and contrast fixated and nonfixated facial silicone implants in different facial sites. A narrative review of the topic of facial-implant stabilization using the PubMed database inclusion criteria included articles that discussed the topic of facial implants, were published in English, and included critical information such as the location of the implant, type of stabilization, follow-up periods, and complications. A total of 11 studies were included. Of these, two were prospective clinical studies, three were case series, and the remaining six were retrospective clinical studies. The studies were published between 1995 and 2018. The sample size varied from 2 to 601 cases. Stabilization includes suturing, monocortical screws, or no stabilization. Complications were reported in most of these studies, including asymmetry, bone resorption or erosion, displacement, dissatisfaction, edema, hematoma, infection, mucosal irritation, pain, and paresthesia. The follow-up period ranged from one month to 17 years. Despite the varied settings of these studies, silicone facial implant complications were reported in both fixated and nonfixated implants, with a lack of significant differences between fixated and nonfixated facial silicone implants regarding the method of fixation.
PubMed: 36874350
DOI: 10.7759/cureus.34524 -
Frontiers in Microbiology 2020The Arctic Ocean is the smallest ocean on Earth, yet estimated to play a substantial role as a global carbon sink. As climate change is rapidly changing fundamental... (Review)
Review
The Arctic Ocean is the smallest ocean on Earth, yet estimated to play a substantial role as a global carbon sink. As climate change is rapidly changing fundamental components of the Arctic, it is of local and global importance to understand and predict consequences for its carbon dynamics. Primary production in the Arctic Ocean is often nitrogen-limited, and this is predicted to increase in some regions. It is therefore of critical interest that biological nitrogen fixation, a process where some bacteria and archaea termed diazotrophs convert nitrogen gas to bioavailable ammonia, has now been detected in the Arctic Ocean. Several studies report diverse and active diazotrophs on various temporal and spatial scales across the Arctic Ocean. Their ecology and biogeochemical impact remain poorly known, and nitrogen fixation is so far absent from models of primary production in the Arctic Ocean. The composition of the diazotroph community appears distinct from other oceans - challenging paradigms of function and regulation of nitrogen fixation. There is evidence of both symbiotic cyanobacterial nitrogen fixation and heterotrophic diazotrophy, but large regions are not yet sampled, and the sparse quantitative data hamper conclusive insights. Hence, it remains to be determined to what extent nitrogen fixation represents a hitherto overlooked source of new nitrogen to consider when predicting future productivity of the Arctic Ocean. Here, we discuss current knowledge on diazotroph distribution, composition, and activity in pelagic and sea ice-associated environments of the Arctic Ocean. Based on this, we identify gaps and outline pertinent research questions in the context of a climate change-influenced Arctic Ocean - with the aim of guiding and encouraging future research on nitrogen fixation in this region.
PubMed: 33391213
DOI: 10.3389/fmicb.2020.596426 -
Frontiers in Molecular Biosciences 2022Precision medicine is "an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for... (Review)
Review
Precision medicine is "an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person." Among many medical specialists involved in precision medicine, the pathologists play an important and key role in the implementation and development of molecular tests that are in the center of decision of many therapeutic choices. Besides many laboratory procedures directly involved in the molecular tests, is fundamental to guarantee that tissues and cells collected for analysis be managed correctly before the DNA/RNA extraction. In this paper we explore the pivotal and interconnected points that can influence molecular studies, such as pre-analytical issues (fixation and decalcification); diagnosis and material selection, including the calculation of nuclei neoplastic fraction. The standardization of sample processing and morphological control ensures the accuracy of the diagnosis. Tissue or cytological samples constitutes the main foundation for the determination of biomarkers and development of druggable targets. Pathology and precision oncology still have a long way to go in terms of research and clinical practice: improving the accuracy and dissemination of molecular tests, learning in molecular tumor boards for advanced disease, and knowledge about early disease. Precision medicine needs pathology to be precise.
PubMed: 36387281
DOI: 10.3389/fmolb.2022.983102